Department of Pediatrics, University of Nebraska Medical Center, Omaha, NE, USA.
Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, NE, USA.
FASEB J. 2020 May;34(5):6351-6368. doi: 10.1096/fj.201902260R. Epub 2020 Mar 13.
Site-directed mutagenesis allows the generation of novel DNA sequences that can be used for a variety of important applications such as the functional analysis of genetic variants. To overcome the limitations of existing site-directed mutagenesis approaches, we explored in vivo DNA gap repair. We found that site-specific mutations in plasmid DNA can be generated in Escherichia coli using mutant single-stranded oligonucleotides to target PCR-derived linear double-stranded plasmid DNA. We called this method DeGeRing, and we characterized its advantages, including non-biased multiplex mutagenesis, over existing site-directed mutagenesis methods such as recombineering (recombination-mediated genetic engineering), single DNA break repair (SDBR, introduced by W. Mandecki), and QuikChange (Agilent Technologies, La Jolla, CA). We determined the efficiency of DeGeRing to induce site-directed mutations with and without a phenotype in three K-12 E coli strains using multiple single-stranded oligonucleotides containing homological and heterological parts of various sizes. Virtual lack of background made the isolation of mutants with frequencies up to 10 unnecessary. Our data show that endogenous DNA gap repair in E coli supports efficient multiplex site-directed mutagenesis. DeGeRing might facilitate the generation of mutant DNA sequences for protein engineering and the functional analysis of genetic variants in reverse genetics.
定点突变允许生成新的 DNA 序列，可用于各种重要的应用，如遗传变异的功能分析。为了克服现有定点突变方法的局限性，我们探索了体内 DNA 缺口修复。我们发现，使用突变的单链寡核苷酸靶向源自 PCR 的线性双链质粒 DNA，可以在大肠杆菌中生成质粒 DNA 的定点突变。我们将这种方法称为 DeGeRing，并对其优势进行了描述，包括非偏向性多重诱变，优于现有的定点突变方法，如重组（重组介导的基因工程）、单 DNA 断裂修复（SDBR，由 W. Mandecki 引入）和 QuikChange（加利福尼亚州拉霍亚的安捷伦科技公司）。我们使用包含同源和异源部分的多种大小的单链寡核苷酸，在三个 K-12 大肠杆菌菌株中确定了 DeGeRing 在有表型和无表型情况下诱导定点突变的效率。几乎不存在背景，使得无需以高达 10 的频率分离突变体。我们的数据表明，大肠杆菌中的内源性 DNA 缺口修复支持高效的多重定点突变。DeGeRing 可能有助于生成用于蛋白质工程和反向遗传学中遗传变异功能分析的突变 DNA 序列。
